Hla-a* 1101-restricted wt1 peptide and pharmaceutical composition comprising the same

ABSTRACT

An HLA-A*1101-restricted WT1 peptide, specifically, a peptide comprising an amino acid sequence consisting of 9 contiguous amino acids from a WT1 protein, wherein the peptide has an ability to bind to an HLA-A*1101 molecule, and has an ability to induce a CTL is described. A peptide dimer having an ability to bind to an HLA-A*1101 molecule and having an ability to induce a CTL, in which two peptide monomers each comprising an amino acid sequence consisting of 9 contiguous amino acids from a WT1 protein and comprising at least one cysteine residue are bound to each other through a disulfide bond is also described. Furthermore, a polynucleotide encoding the peptide, a pharmaceutical composition for the treatment and/or prevention of a cancer comprising the same and the like are provided.

TECHNICAL FIELD

The present invention relates to an HLA-A*1101-restricted WT1 peptide,specifically, a peptide comprising an amino acid sequence consisting of9 contiguous amino acids from a WT1 protein, wherein the peptide has anability to bind to an HLA-A*1101 molecule, and has an ability to inducea CTL. The present invention also relates to a peptide dimer having anability to bind to an HLA-A*1101 molecule, and having an ability toinduce a CTL, wherein two peptide monomers each comprising an amino acidsequence consisting of 9 contiguous amino acids from a WT1 protein andcomprising at least one cysteine residue are bound to each other througha disulfide bond. Furthermore, the present invention relates to apolynucleotide encoding the peptide, a pharmaceutical composition forthe treatment and/or prevention of a cancer comprising the same and thelike.

BACKGROUND

WT1 gene (Wilms' tumor 1 gene) was identified as a gene responsible forWilms tumor which is a renal cancer in children (Non-patent Documents 1and 2). WT1 is a transcription factor having a zinc finger structure. Atthe beginning, the WT1 gene was considered to be a tumor suppressorgene. However, subsequent studies (Non-patent Documents 3, 4, 5 and 6)showed that the WT1 gene rather functions as an oncogene inhematopoietic tumors and solid cancers.

The WT1 gene is expressed at high levels in many types of malignanttumors. Then, it has been examined whether or not the WT1 gene productfree of mutations, which is an autologous protein, has immunogenicity ina living body. The results revealed that the protein derived from theWT1 gene which is expressed at high levels in tumor cells is fragmentedthrough intracellular processing, the resulting peptides form complexeswith MHC class I molecules, and the complexes are presented on thesurfaces of cells, and that CTLs recognizing such complexes can beinduced by peptide vaccination (Non-patent Documents 7, 8 and 9). It wasalso shown that in a mouse immunized with a WT1 peptide or a WT1 cDNA,transplanted tumor cells expressing a WT1 gene are rejected with a highprobability (Non-patent Documents 7 and 10), while normal tissuesexpressing physiologically the WT1 gene are not damaged by the inducedCTLs (Non-patent Document 7). It was shown in in vitro experiments usinghuman cells that when Db126 peptide or WH187 peptide (amino acids187-195 of SEQ ID No: 1, SLGEQQYSV) having a high ability to bind to anHLA-A*0201 molecule, which is one of human MHC class I molecules, isused to stimulate human peripheral blood mononuclear cells havingHLA-A*0201, WT1-specific CTLs are induced, the induced CTLs have acytotoxic activity specific for tumor cells expressing endogenously aWT1 gene at high levels, and the cytotoxic activity of such CTLs isHLA-A2-restricted (Non-patent Document 11). It was shown in in vitroexperiments in human cells using WT1 peptide that matches HLA-A*2402(which is found most frequently in Japanese people among HLA-A alleles)(WT1235; amino acids 235-243 of SEQ ID No: 1, CMTWNQMNL) thatWT1-specific CTLs (TAK-1) are induced (Non-patent Document 12), and theinduced CTLs do not suppress the colony-forming activity of normalhematopoietic stem cells which partially express physiologically a WT1gene (Non-patent Documents 12 and 13). These reports strongly suggestthat not only in mice but also in human beings, WT1-specific CTLs can beinduced, such CTLs have a cytotoxic activity against tumor cellsexpressing a WT1 gene at high levels, but do not have a cytotoxicactivity against normal cells expressing physiologically a WT1 gene(Non-patent Documents 7, 10, 11, 12 and 13).

The WT1 gene product is present as a nuclear protein, and is processedby proteasomes in cytoplasm to be fragmented into peptides. Thefragmented peptides are transported into endoplasmic reticulum lumen byTAP (transporter associated with antigen processing) molecules, formcomplexes with MHC class I molecules, and are presented on the surfacesof cells. WT1-specific CTLs are induced as a result of recognition ofWT1 peptide-MHC class I molecule complexes by CTL precursor cells viaTCR, thereby exerting a cytotoxic effect on tumor cells presenting a WT1gene product through MHC class I molecules (Non-patent Documents 7, 8and 9). Then, it is required at least that a WT1 peptide used in cancerimmunotherapy targeting a WT1 gene product is in the form that binds toan MHC class I molecule in a living body. However, MHC class I moleculesare diverse and amino acid sequences of the WT1 peptides binding torespective MHC class I molecules are different from each other.Therefore, it is required to provide a peptide matching each subtype ofMHC class I. However, only HLA-A*2402 molecule-, HLA-A*0201 molecule-,HLA-A*2601 molecule- and HLA-A*3303 molecule-restricted peptides areknown as HLA molecule-restricted WT1 peptides to date (Patent Document1, Non-patent Document 11, Patent Document 2 and Patent Document 3,respectively). Thus, there is a need to find an HLA-A*1101-restrictedWT1 peptide.

-   Patent Document 1: WO 2003/106682-   Patent Document 2: WO 2005/095598-   Patent Document 3: Japanese Patent Application No. 2006-45287-   Non-patent Document 1: Daniel A. Haber et al., Cell. 1990 Jun. 29;    61(7):1257-69.-   Non-patent Document 2: Call K M et al., Cell. 1990 Feb. 9;    60(3):509-20.-   Non-patent Document 3: Menke A L et al., Int Rev Cytol. 1998;    181:151-212. Review.-   Non-patent Document 4: Yamagami T et al., Blood. 1996 Apr. 1;    87(7):2878-84.-   Non-patent Document 5: Inoue K et al., Blood. 1998 Apr. 15;    91(8):2969-76.-   Non-patent Document 6: Tsuboi A et al., Leuk Res. 1999 May;    23(5):499-505.-   Non-patent Document 7: Oka Y et al., J Immunol. 2000 Feb. 15;    164(4):1873-80.-   Non-patent Document 8: Melief C J et al., Immunol Rev. 1995 June;    145:167-77.-   Non-patent Document 9: Ritz J, J Clin Oncol. 1994 February;    12(2):237-8.-   Non-patent Document 10: Tsuboi A et al., J Clin Immunol. 2000 May;    20(3):195-202.-   Non-patent Document 11: Oka Y et al., Immunogenetics. 2000 February;    51(2):99-107.-   Non-patent Document 12: Ohminami H et al., Blood. 2000 Jan. 1;    95(1):286-93.-   Non-patent Document 13: Gao L et al., Blood. 2000 Apr. 1;    95(7):2198-203.

DISCLOSURE OF INVENTION Problems to be Solved by the Invention

The problems to be solved by the present invention are to provide apeptide that is an HLA-A*1101 molecule-restricted and comprises an aminoacid sequence from a WT1 protein, and a polynucleotide encoding thesame, as well as a pharmaceutical composition for the treatment and/orprevention of a cancer, comprising the same, and the like.

Means to Solve the Problems

As a result of intensive studies in view of the situation as describedabove, the present inventor has found that among peptides eachcomprising an amino acid sequence consisting of 9 contiguous amino acidsfrom a WT1 protein, peptides each having an ability to bind to anHLA-A*1101 molecule can induce a WT1-specific CTL with a high rate.Thus, the present invention has been completed.

The present invention provides:

(1) a peptide comprising an amino acid sequence consisting of 9contiguous amino acids from a WT1 protein, wherein the peptide has anability to bind to an HLA-A*1101 molecule, and has an ability to inducea CTL;

(2) the peptide according to (1), wherein the amino acid at position 9of the amino acid sequence is Lys or Arg;

(3) the peptide according to (1), wherein the amino acid sequence isselected from the group consisting of:

(SEQ ID No: 2) Ala Ala Gly Ser Ser Ser Ser Val Lys, (SEQ ID No: 3)Pro Ile Leu Cys Gly Ala Gln Tyr Arg, (SEQ ID No: 4)Arg Ser Ala Ser Glu Thr Ser Glu Lys, (SEQ ID No: 5)Ser Ala Ser Glu Thr Set Glu Lys Arg, (SEQ ID No: 6)Ser His Leu Gln Met His Set Arg Lys, (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys, (SEQ ID No: 8)Lys Thr Cys Gln Arg Lys Phe Ser Arg, (SEQ ID No: 9)Ser Cys Arg Trp Pro Ser Cys Gln Lys, and (SEQ ID No: 10)Asn Met His Gln Arg Asn Met Thr Lys;

(4) the peptide according to (3), wherein the amino acid sequence is AlaAla Gly Ser Ser Ser Ser Val Lys (SEQ ID No: 2);

(5) a peptide dimer having an ability to bind to an HLA-A*1101 moleculeand having an ability to induce a CTL, in which two peptide monomerseach comprising an amino acid sequence consisting of 9 contiguous aminoacids from a WT1 protein, and comprising at least one cysteine residueare bound to each other through a disulfide bond;

(6) the peptide dimer according to (5), wherein the amino acid sequenceof the peptide monomer is selected from the group consisting of:

(SEQ ID No: 3) Pro Ile Leu Cys Gly Ala Gln Tyr Arg, (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys, (SEQ ID No: 8)Lys Thr Cys Gln Arg Lys Phe Ser Arg, and (SEQ ID No: 9)Ser Cys Arg Trp Pro Ser Cys Gln Lys;

(7) a pharmaceutical composition for the treatment or prevention of acancer, comprising the peptide according to (1) and/or the peptide dimeraccording to (5);

(8) a method for the treatment or prevention of a cancer, comprisingadministering an effective amount of the peptide according to (1) and/orthe peptide dimer according to (5) to an HLA-A*1101-positive subject;

(9) a polynucleotide encoding the peptide according to (1);

(10) an expression vector comprising the polynucleotide according to(9);

(11) a pharmaceutical composition for the treatment or prevention of acancer, comprising the polynucleotide according to (9) or the vectoraccording to (10);

(12) a method for the treatment or prevention of a cancer, comprisingadministering an effective amount of the polynucleotide according to (9)or the vector according to (10) to an HLA-A*1101-positive subject;

(13) a WT1-specific CTL, which is induced by the peptide according to(1) and/or the peptide dimer according to (5);

(14) a method for the induction of a WT1-specific CTL, comprisingculturing a peripheral blood mononuclear cell in the presence of thepeptide according to (1) and/or the peptide dimer according to (5) toinduce the WT1-specific CTL from the peripheral blood mononuclear cell;

(15) a kit for the induction of a WT1-specific CTL, comprising thepeptide according to (1) and/or the peptide dimer according to (5) as anessential component;

(16) an antigen-presenting cell presenting a WT1 peptide, which isinduced by the peptide according to (1) and/or the peptide dimeraccording to (5);

(17) a method for the induction of an antigen-presenting cell presentinga WT1 peptide, comprising culturing an immature antigen-presenting cellin the presence of the peptide according to (1) and/or the peptide dimeraccording to (5) to induce the antigen-presenting cell presenting a WT1peptide from the immature antigen-presenting cell;

(18) a kit for the induction of an antigen-presenting cell presenting aWT1 peptide, comprising the peptide according to (1) and/or the peptidedimer according to (5) as an essential component; and

(19) a method for the diagnosis of a cancer, comprising using the CTLaccording to (13) or the antigen-presenting cell according to (16).

Effects of the Invention

The present invention provides a peptide that is HLA-A*1101-restrictedand comprises an amino acid sequence consisting of 9 contiguous aminoacids from a WT1 protein, and a polynucleotide encoding the same, aswell as a pharmaceutical composition for the treatment and/or preventionof a cancer, comprising the same, and the like. Therefore, it ispossible to induce in vivo and in vitro WT1-specific CTLs in subjectshaving HLA-A*1101. Because the rate of HLA-A*1101-positive in Japanesepeople is high (about 17.9%), WT1-specific CTLs can be induced in a widerange of subjects.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 represents the cytotoxic activity of the CTL induced with WT1₂₅₁.

FIG. 2 represents the cytotoxic activity of the CTL induced with WT1₂₇₉.

FIG. 3 represents the cytotoxic activity of the CTL induced with WT1₃₁₂.

FIG. 4 represents the cytotoxic activity of the CTL induced with WT1₃₁₃.

FIG. 5 represents the cytotoxic activity of the CTL induced with WT1₃₃₈.

FIG. 6 represents the cytotoxic activity of the CTL induced with WT1₃₇₈.

FIG. 7 represents the cytotoxic activity of the CTL induced with WT1₃₈₆.

FIG. 8 represents the cytotoxic activity of the CTL induced with WT1₄₁₃.

FIG. 9 represents the cytotoxic activity of the CTL induced with WT1₄₃₆.

FIG. 10 represents the cytotoxic activity of the CTL induced with WT1₃₇₈peptide (a, b and c represent the cytotoxic activities observed usingPBMCs from HLA-A*1101-positive healthy donors 1, 2 and 3, respectively).

FIG. 11 represents the cytotoxic activity of the CTL induced with WT1₃₇₈peptide dimer (a and b represent the cytotoxic activities observed usingPBMCs from HLA-A*1101-positive healthy donors 1 and 2, respectively).

FIG. 12 represents the cytotoxic activity of the CTL induced withmodified WT1₃₇₈ peptide (G→I) (a, b and c represent the cytotoxicactivities observed using PBMCs from HLA-A*101-positive healthy donors1, 2 and 3, respectively).

FIG. 13 represents the cytotoxic activity of the CTL induced withmodified WT1₃₇₈ peptide (G→V) (a, b and c represent the cytotoxicactivities observed using PBMCs from HLA-A*1101-positive healthy donors1, 2 and 3, respectively).

FIG. 14 represents the cytotoxic activity of the CTL induced with WT1₃₇₉peptide (a, b and c represent the cytotoxic activities observed usingPBMCs from HLA-A*1101-positive healthy donors 1, 2 and 3, respectively).

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to a peptide comprising anamino acid sequence consisting of 9 contiguous amino acids from a WT1protein, wherein the peptide has an ability to bind to an HLA-A*1101molecule, and has an ability to induce a CTL (herein also referred to asa “WT1 peptide”). The amino acid sequence of the human WT1 protein isshown in SEQ ID No: 1. The peptide of the present invention comprises anamino acid sequence consisting of 9 contiguous amino acids in the aminoacid sequence shown in SEQ ID No: 1. When the peptide of the presentinvention comprises an amino acid sequence comprising cystein(s) such asthe amino acid sequence of SEQ ID No: 3, 7, 8 or 9 as described below,the stability may be increased by substituting the cystein(s) in theamino acid sequence with another substance such as another amino acid(for example, serine, alanine and α-aminobutyric acid) or by modifyingthe SH group of the cystein(s) with a protecting group known in the art(for example, carboxymethyl group or pyridylethyl group). The peptide ofthe present invention is a cancer antigen peptide that can induce a CTLas a result of presentation, by an antigen-presenting cell, of a peptidegenerated by processing the peptide of the present invention in a cell.

As described above, it is an object of the present invention to obtainan HLA-A*1101-restricted peptide. Thus, the peptide of the presentinvention has an ability to bind to an HLA-A*1101 molecule. The abilityto bind can be determined by a method known in the art. Examples of suchmethods include a computer-based method such as Rankpep, BIMAS orSYFPEITHI, and a competitive binding test with a known peptide having anability to bind to an HLA-A*1101 molecule. For example, the determinedability to bind can be compared with that of a knownHLA-A*1101-restricted peptide to judge whether or not the peptide of thepresent invention has an ability to bind. Examples of peptides having anability to bind according to the present invention include a peptide ofwhich the affinity score to an HLA-A*1101 molecule as determined by themethod described in example 1 is 4 or more, preferably 5 or more, morepreferably 6 or more.

The peptide of the present invention further has an ability to induce aCTL. The WT1 gene is expressed in its native form at high levels, forexample, in hematopoietic tumors such as leukemia, myelodysplasticsyndrome, multiple myeloma or malignant lymphoma and solid cancers suchas gastric cancer, colon cancer, lung cancer, breast cancer, germ cellcancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer,uterine cancer, cervical cancer or ovarian cancer. Therefore, thepeptide of the present invention can induce a CTL with a high rate in asubject suffering from such a disease. The ability to induce a CTLrefers to an ability to induce a CTL in vivo or in vitro. Such anability can be determined using a general method such as a method inwhich a cytotoxic activity of a CTL is determined using a Cr-releaseassay.

The peptide of the present invention may have Lys or Arg at position 9of the amino acid sequence. It is considered that by having such anamino acid, the ability of the peptide to bind to an HLA-A*1101 moleculebecomes higher.

The amino acid sequence consisting of 9 amino acids comprised in thepeptide of the present invention is preferably, Ala Ala Gly Ser Ser SerSer Val Lys (SEQ ID No: 2), Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ IDNo: 3), Arg Ser Ala Ser Glu Thr Ser Glu Lys (SEQ ID No: 4), Ser Ala SerGlu Thr Ser Glu Lys Arg (SEQ ID No: 5), Ser His Leu Gln Met His Ser ArgLys (SEQ ID No: 6), Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ ID No: 7),Lys Thr Cys Gln Arg Lys Phe Ser Arg (SEQ ID No: 8), Ser Cys Arg Trp ProSer Cys Gin Lys (SEQ ID No: 9) or Asn Met His Gln Arg Asn Met Thr Lys(SEQ ID No: 10). Most preferably, it is Thr Gly Val Lys Pro Phe Gln CysLys (SEQ ID No: 7). Furthermore, it may have a substitution of one toseveral, preferably one to five amino acids with other amino acids inthe nine amino acids of any of SEQ ID Nos: 2-10. Any one of the 9 aminoacids or other substituted amino acids may be appropriately modified. Inany cases, the peptide of the present invention retains an ability tobind to an HLA-A*1101 molecule.

As described above, the peptide of the present invention may be any oneas long as it comprises an amino acid sequence that is derived from aWT1 protein and consists of 9 contiguous amino acids. Thus, the peptideof the present invention may be, for example, a peptide consisting ofonly the amino acid sequence shown in any of SEQ ID Nos: 2-10, or a WT1protein or a part thereof comprising the amino acid sequence shown inany of SEQ ID Nos: 2-10. The number of amino acids comprised in thepeptide of the present invention is not specifically limited, and thenumber is, for example, 9-500, 9-300, 9-200, 9-100, 9-50, 9-30 and 9-12amino acids. Various substances may be attached at the N-terminus and/orthe C-terminus of the amino acid sequence consisting of 9 contiguousamino acids in the peptide of the present invention. For example, anamino acid, a peptide or an analog thereof may be attached. If such asubstance is attached to the peptide of the present invention, thesubstance can be processed, for example, by an enzyme in a living bodyor through a process such as intracellular processing, and finally theamino acid sequence consisting of 9 contiguous amino acids can beproduced and presented as a complex with an HLA-A*1101 molecule on thesurface of a cell, thereby resulting in the effect of inducing a CTL.The substance may be a substance that modulates the solubility of thepeptide of the present invention, or increases its stability (resistanceto protease, etc.). Alternatively, it may be a substance that deliversthe peptide of the present invention specifically, for example, to agiven tissue or organ, or it may have an action to increase theefficiency of uptake by an antigen-presenting cell or the like. Thesubstance may be a substance that increases an ability to induce a CTL,such as a helper peptide or the like.

The peptide of the present invention can be synthesized by methodsgenerally used in the art or modifications thereof. Such methods aredescribed, for example, in Peptide Synthesis, Interscience, New York,1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976;Peptide-Gosei, Maruzen Co., Ltd., 1975; Peptide-Gosei No Kiso To Jikken,Maruzen Co., Ltd., 1985; and Iyakuhin No Kaihatsu (Zoku), Vol. 14,Peptide-Gosei, Hirokawa—Book store, 1991.

The peptide of the present invention can also be prepared using geneticengineering techniques based on the information about the nucleotidesequence that encodes the peptide of the present invention. Such geneticengineering techniques are well known to a skilled person in the art.

In a further aspect, the present invention relates to a peptide dimerhaving an ability to bind to an HLA-A*1101 molecule and having anability to induce a CTL, in which two peptide monomers each comprisingan amino acid sequence consisting of 9 contiguous amino acids from a WT1protein and comprising at least one cystein residue are bound to eachother though a disulfide bond (hereinafter also referred to as a “WT1peptide dimer”). The stability of the peptide dimer of the presentinvention is increased as compared with that of the peptide monomer byforming a dimer. The peptide dimer of the present invention is a tumorantigen peptide dimer that can induce a CTL as a result of presentation,by an antigen-presenting cell, of a peptide generated by processing thepeptide of the present invention in a cell.

The peptide dimer of the present invention is formed by binding twopeptide monomers through a disulfide bond between cystein residuespresent in the monomers. Thus, each of the peptide monomers comprised inthe WT1 peptide dimer of the present invention is the WT1 peptide asdescribed above and comprises at least one cystein residue. The WT1peptide dimer of the present invention may be a homodimer or aheterodimer.

In the WT1 peptide dimer of the present invention, the amino acidsequence comprised by the peptide monomer comprises is preferably, ProIle Leu Cys Gly Ala Gin Tyr Arg (SEQ ID No: 3), Thr Gly Val Lys Pro PheGln Cys Lys (SEQ ID No: 7), Lys Thr Cys Gin Arg Lys Phe Ser Arg (SEQ IDNo: 8) or Ser Cys Arg Trp Pro Ser Cys Gln Lys (SEQ ID No: 9). Mostpreferably, it is Thr Gly Val Lys Pro Phe Gln Cys Lys (SEQ ID No: 7).

The WT1 peptide dimer of the preset invention can be prepared using amethod known in the art. For example, if the peptide monomers compriseone pair of cystein residues, the WT1 peptide dimer of the presentinvention can be prepared, for example, by removing all the protectinggroups including the ones on the cystein side chains, and thensubjecting the resulting monomer solution to air-oxidation underalkaline conditions, or adding an oxidant under alkaline or acidicconditions to form a disulfide bond. Examples of the oxidants includeiodine, dimethylsulfoxide (DMSO) and potassium ferricyanide.

When each of the peptide monomers comprises two or more cysteinresidues, the WT1 peptide dimer of the present invention can also beprepared by the method as described above. In this case, isomers areobtained due to different types of disulfide bonds. Alternatively, theWT1 peptide dimer of the present invention can be prepared by selectinga combination of protecting groups for cystein side chains. Examples ofthe combinations of the protecting groups include combinations of MeBzl(methylbenzyl) group and Acm (acetamidemethyl) group, Trt (trityl) groupand Acm group, Npys (3-nitro-2-pyridylthio) group and Acm group, andS-Bu-t (S-tert-butyl) group and Acm group. For example, in the case ofthe combination of MeBzl group and Acm group, the WT1 peptide dimer canbe prepared by removing protecting groups other than the MeBzl group andthe protecting group on the cystein side chain, subjecting the resultingmonomer solution to air-oxidation to form a disulfide bond between theprotected cystein residues, and then deprotecting and oxidizing usingiodine to form a disulfide bond between the cystein residues previouslyprotected by Acm.

In another aspect, the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of a cancer comprising theHLA-A*1101-restricted WT1 peptide and/or the WT1 peptide dimer. The WT1gene is expressed at high levels in various cancers and tumors includinghematopoietic tumors such as leukemia, myelodysplastic syndrome,multiple myeloma or malignant lymphoma and solid cancers such as gastriccancer, colon cancer, lung cancer, breast cancer, germ cell cancer,hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterinecancer, cervical cancer or ovarian cancer. Therefore, the pharmaceuticalcomposition of the present invention can be used for the treatment orprevention of a cancer. When the pharmaceutical composition of thepresent invention is administered to an HLA-A*1101-positive subject,WT1-specific CTLs are induced by the HLA-A*1101-restricted WT1 peptideor the WT1 peptide dimer comprised in the pharmaceutical composition,and cancer cells in the subject are damaged by such CTLs.

The pharmaceutical composition of the present invention may comprise inaddition to the HLA-A*1101-restricted WT1 peptide and/or the WT1 peptidedimer as an active ingredient, for example, a carrier, an excipient orthe like. The HLA-A*1101-restricted WT1 peptide or the WT1 peptide dimercomprised in the pharmaceutical composition of the present inventioninduces a WT1-specific CTL. Thus, the pharmaceutical composition of thepresent invention may comprise an appropriate adjuvant, or may beadministered together with an appropriate adjuvant in order to enhancethe induction efficiency. Examples of preferable adjuvants include, butare not limited to, complete or incomplete Freund's adjuvant andaluminum hydroxide.

The method of the administration of the pharmaceutical composition ofthe present invention can be appropriately selected depending onconditions such as the type of disease, the condition of the subject orthe target site. Examples of such methods include, but are not limitedto, intradermal administration, subcutaneous administration,intramuscular administration, intravenous administration, nasaladministration and oral administration. The amount of the peptide or thepeptide dimer comprised in the pharmaceutical composition of the presentinvention, as well as the dosage form, the number of times of theadministration and the like of the pharmaceutical composition of thepresent invention can be appropriately selected depending on conditionssuch as the type of disease, the condition of the subject or the targetsite. The single dose of the peptide is usually, 0.0001 mg-1000 mg,preferably, 0.001 mg-1000 mg.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering aneffective amount of the WT1 peptide and/or the WT1 peptide dimer to anHLA-A*1101-positive subject. The cancer to be treated or prevented maybe any one, and examples thereof include hematopoietic tumors such asleukemia, myelodysplastic syndrome, multiple myeloma or malignantlymphoma and solid cancers such as gastric cancer, colon cancer, lungcancer, breast cancer, germ cell cancer, hepatic cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical cancer orovarian cancer.

In a further aspect, the present invention relates to a method for thedetermination of the presence or amount of a WT1-specific CTL in anHLA-A*1101-positive subject, comprising:

(a) reacting a complex of a WT1 peptide and an HLA-A*1101 molecule witha sample from the subject; and(b) determining the presence or amount of a CTL recognizing the complexcontained in the sample. The sample from a subject may be any one aslong as there is a possibility that it contains a lymphocyte. Examplesof the samples include body fluid such as blood or lymph and a tissue.The complex of a WT1 peptide and an HLA-A*1101 molecule may be prepared,for example, as a tetramer or pentamer using a method known to a skilledperson in the art such as biotin-streptavidin method. The presence oramount of the CTL recognizing such a complex can be measured by a methodknown to a skilled person in the art. In this aspect of the presentinvention, the complex may be labeled. A known label such as afluorescent label or a radioactive label can be used as a label.Labeling makes the determination of the presence or amount of a CTL easyand rapid.

Thus, the present invention also provides a composition for thedetermination of the presence or amount of a WT1-specific CTL in anHLA-A*1101-positive subject comprising an HLA-A*1101-restricted WT1peptide.

Furthermore, the present invention provides a kit for the determinationof the presence or amount of a WT1-specific CTL in anHLA-A*1101-positive subject, comprising an HLA-A*1101-restricted WT1peptide.

In a further aspect, the present invention relates to a method for theproduction of a WT1-specific CTL using a complex of a WT1 peptide and anHLA-A*1101 molecule, comprising:

(a) reacting the complex with a sample; and(b) obtaining a CTL recognizing the complex contained in the sample. Thecomplex of a WT1 peptide and an HLA-A*1101 molecule is described above.The sample may be any one as long as there is a possibility that itcontains a lymphocyte. Examples of the samples include a sample from asubject such as blood, and a cell culture. The CTL recognizing thecomplex can be obtained using a method known to a skilled person in theart such as FACS or MACS. The present invention allows to culture theobtained WT1-specific CTL and use it for the treatment or prevention ofvarious cancers.

Thus, the present invention also relates to a WT1-specific CTLobtainable by a method for the production of a WT1-specific CTL using acomplex of a WT1 peptide and an HLA-A*1101 molecule.

In another aspect, the present invention relates to a polynucleotideencoding the HLA-A*1101-restricted WT1 peptide. The polynucleotide ofthe present invention may be DNA or RNA. The base sequence of thepolynucleotide of the present invention can be determined based on theamino acid sequence of the HLA-A*1101-restricted WT1 peptide. Thepolynucleotide can be prepared by a known method for the synthesis ofDNA or RNA (for example, chemical synthetic method), PCR method or thelike.

In another aspect, the present invention relates to an expression vectorcomprising the polynucleotide. The type of the expression vector, thecomprised sequence other than the sequence of the polynucleotide and thelike can be appropriately selected depending on the type of a host intowhich the expression vector of the present invention is introduced, thepurpose of use, or the like. It is possible to treat or preventhematopoietic tumors or solid cancers by administering the expressionvector of the present invention to an HLA-A*1101-positive subject toproduce an HLA-A*1101-restricted WT1 peptide in a living body and inducea WT1-specific CTL, and damaging hematopoietic tumor cells or solidcancer cells in the subject.

In a further aspect, the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of a cancer, comprising thepolynucleotide or the expression vector. The composition, method of theadministration and the like of the pharmaceutical composition of thepresent invention in this aspect are described above.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering aneffective amount of the polynucleotide or the expression vector to anHLA-A*1101-positive subject. Examples of cancers to be treated orprevented include hematopoietic tumors such as leukemia, myelodysplasticsyndrome, multiple myeloma or malignant lymphoma and solid cancers suchas gastric cancer, colon cancer, lung cancer, breast cancer, germ cellcancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer,uterine cancer, cervical cancer or ovarian cancer.

In another aspect, the present invention relates to a cell comprisingthe expression vector. The cell of the present invention can beprepared, for example, by transforming a host cell such as E. coli,yeast, insect cell or animal cell with the expression vector. The methodfor the introduction of the expression vector into a host cell can beappropriately selected from various methods. By culturing thetransformed cell, and recovering and purifying the produced WT1 peptide,the peptide of the present invention can be prepared.

In a further aspect, the present invention relates to a WT1-specificCTL, which is induced by the HLA-A*1101-restricted WT1 peptide and/orthe WT1 peptide dimer. The CTL of the present invention recognizes acomplex of a WT1 peptide and an HLA-A*1101 molecule. Thus, the CTL ofthe present invention can be used to damage specifically a tumor cellpositive for HLA-A*1101 and expressing WT1 at a high level.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering aWT1-specific CTL to an HLA-A*1101-positive subject. The method of theadministration of the WT1-specific CTL can be appropriately selecteddepending on conditions such as the type of the disease, the conditionof the subject or the target site. Examples of such methods include, butare not limited to, intravenous administration, intradermaladministration, subcutaneous administration, intramuscularadministration, nasal administration and oral administration.

In another aspect, the present invention relates to a method for theinduction of a WT1-specific CTL, comprising culturing a peripheral bloodmononuclear cell in the presence of the HLA-A*1101-restricted WT1peptide and/or the WT1 peptide dimer to induce the WT1-specific CTL formthe peripheral blood mononuclear cell. The subject from which theperipheral blood mononuclear cell is derived may be any one as long asit is positive for HLA-A*1101. By culturing the peripheral bloodmononuclear cells in the presence of the HLA-A*1101-restricted WT1peptide and/or the WT1 peptide dimer, WT1-specific CTLs are induced fromCTL precursor cells contained in the peripheral blood mononuclear cells.It is possible to treat or prevent hematopoietic tumors or solid cancersin an HLA-A*1101-positive subject by administering the WT1-specific CTLobtained according to the present invention to the subject.

In another aspect, the present invention relates to a kit for theinduction of a WT1-specific CTL, comprising an HLA-A*1101-restricted WT1peptide and/or the WT1 peptide dimer as an essential component.Preferably, the kit is used in the method for the induction of aWT1-specific CTL. The kit of the present invention may comprise inaddition to the HLA-A*1101-restricted WT1 peptide and/or the WT1 peptidedimer, for example, a means of obtaining a peripheral blood mononuclearcell, an adjuvant, a reaction vessel or the like. In general, aninstruction manual is attached to the kit. By using the kit of thepresent invention, WT1-specific CTLs can be induced efficiently.

In a further aspect, the present invention relates to anantigen-presenting cell (such as a dendritic cell) presenting a WT1peptide through an HLA-A*1101 molecule, which is induced by theHLA-A*1101-restricted WT1 peptide and/or the WT1 peptide dimer. By usingthe antigen-presenting cell of the present invention, WT1-specific CTLsare induced efficiently.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering theantigen-presenting cell presenting a WT1 peptide through an HLA-A*1101molecule to an HLA-A*1101-positive subject. The method of theadministration of the antigen-presenting cell can be appropriatelyselected depending on conditions such as the type of the disease, thecondition of the subject or the target site. Examples of such methodsinclude, but are not limited to, intravenous administration, intradermaladministration, subcutaneous administration, intramuscularadministration, nasal administration and oral administration.

In another aspect, the present invention relates to a method for theinduction of an antigen-presenting cell presenting a WT1 peptide throughan HLA-A*1101 molecule, comprising culturing an immatureantigen-presenting cell in the presence of the HLA-A*1101-restricted WT1peptide and/or the WT1 peptide dimer to induce the antigen-presentingcell presenting a WT1 peptide through an HLA-A*1101 molecule from theimmature antigen-presenting cell. The immature antigen-presenting cellrefers a cell such as an immature dendritic cell that can be maturedinto an antigen-presenting cell. A subject from which the immatureantigen-presenting cell is derived may be any one as long as it ispositive for HLA-A*1101. Because the immature antigen-presenting cellsare contained, for example, in peripheral blood mononuclear cells, suchcells may be cultured in the presence of the WT1 peptide and/or the WT1peptide diemr.

In another aspect, the present invention relates to a kit for theinduction of an antigen-presenting cell presenting a WT1 peptide throughan HLA-A*1101 molecule, comprising the HLA-A*1101-restricted WT1 peptideand/or the WT1 peptide dimer as an essential component. Preferably, thekit is used in the method for the induction of an antigen-presentingcell. Another component to be comprised in the kit of the presentinvention and the like are described above. The kit of the presentinvention can be used to induce efficiently an antigen-presenting cellpresenting a WT1 peptide through an HLA-A*1101 molecule.

In another aspect, the present invention relates to an antibody againstan HLA-A*1101-restricted WT1 peptide or a polynucleotide encoding thepeptide. The antibody of the present invention may be a polyclonalantibody or monoclonal antibody.

In a further aspect, the present invention relates to a method for thediagnosis of a cancer, comprising using the WT1-specific CTL, theantigen-presenting cell presenting a WT1 peptide through an HLA-A*1101molecule, or the antibody against an HLA-A-restricted WT1 peptide or apolynucleotide encoding the peptide. Preferably, the WT1-specific CTL isused in the method for the diagnosis of the present invention. Forexample, it is possible to diagnose a cancer by incubating the CTL, theantigen-presenting cell or the antibody with a sample from anHLA-A*1101-positive subject, or administering it to anHLA-A*1101-positive subject, and determining, for example, the position,site or amount thereof. The CTL, the antigen-presenting cell or theantibody may be labeled. By attaching a label, the method for thediagnosis of the present invention can be practiced efficiently.

EXAMPLES

The following examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Example 1 Selection of WT1 Peptide

RANKPEP (http://bio.dfci.harvard.edu/Tools/rankpep.html) was used toselect WT1₂₅₁, WT1₂₇₉, WT1₃₁₂, WT1₃₁₃, WT1₃₃₈, WT1₃₇₈, WT1₃₈₆, WT1₄₁₅and WT1₄₃₆ having a high ability to bind to an HLA-A*1101 moleculederived from peptides from a WT1 protein (SEQ ID No: 1). Amino acidsequences, amino acid numbers in SEQ ID No: 1 and affinity scores to anHLA-A*1101 molecule of these peptides are shown in Table 1.

TABLE 1 Amino acid Amino acid Affinity Peptide Number sequence scoreWT1₂₅₁ 251-259 AAGSSSSVK 15.18 (SEQ ID No: 2) WT1₂₇₉ 279-287 PILCGAQYR11.47 (SEQ ID No: 3) WT1₃₁₂ 312-320 RSASETSEK 14.96 (SEQ ID No: 4)WT1₃₁₃ 313-321 SASETSEKR 6.87 (SEQ ID No: 5) WT1₃₃₈ 338-346 SHLQMNSRK13.72 (SEQ ID No: 6) WT1₃₇₈ 378-386 TGVKPFQCK 11.33 (SEQ ID No: 7)WT1₃₆₆ 386-394 KTCQRKFSR 13.82 (SEQ ID No: 8) WT1₄₁₅ 415-423 SCRWPSCQK10.29 (SEQ ID No: 9) WT1₄₃₆ 436-444 NMHQRNMTK 14.19 (SEQ ID No: 10)

Preparation of B-LCL Cell

Peripheral blood mononuclear cells (PBMCs) were separated byFicoll-Hypaque gradient density centrifugation method from peripheralblood that had been collected from an HLA-A*1101-positive healthy donor.The PBMCs were then seeded to a 24-well cell culture plate at thedensity of about 1×10⁷ in RPMI 1640 medium containing 10% FCS, and aculture supernatant of B95-8 cells (cells producing EB virus) wereadded. They were cultured at 37° C. with 5% CO₂ for about 1 month. B-LCLcells transformed with EB virus, which are B-cell tumor cells, wereobtained. It was confirmed that the resulting B-LCL cells did notexpress WTi gene. B-LCL cells were pulsed by incubating them with 20μg/ml of WT1₂₅₁, WT1₂₇₉, WT1₃₁₂, WT1₃₁₃, WT1₃₃₈, WT1₃₇₈, WT1₃₈₆, WT1₄₁₅or WT1₄₃₆ for 2 hours, and irradiated with 80 Gy of radiation. Theresulting B-LCL cells (hereinafter referred to as B-LCL cells pulsedwith a WT1 peptide) were used as antigen-presenting cells for thefollowing experiments.

Induction of WT1-Specific CTL

3×10⁶ of autologous PBMCs were cultured in a 24-well cell culture platein complete medium (45% RPMI, 45% AMI-V medium and 10% human AB serum)containing 20 μg/ml of WT1₂₅₁, WT1₂₇₉, WT1₃₁₂, WT1₃₁₃, WT1₃₃₈, WT1₃₇₈,WT1₃₈₆, WT1₄₁₅ or WT1₄₃₆ at 37° C. with 5% CO₂ for 1 week to obtainresponding cells. 2×10⁶ of the resulting responding cells werecocultured with 1×10⁶ of the B-LCL cells pulsed with the same WT1peptide in complete medium for 1 week (first stimulation). The PBMCswere coculutured with the B-LCL cells pulsed with the WT1 peptide threemore times (second to fourth stimulations) under the conditions underwhich 20 IU/ml (final concentration) of IL2 was added as follows: secondstimulation: two times every other day from 3 days after the initiationof stimulation; third and fourth stimulations: three times at intervalsof one day from the day after the initiation of stimulation. Theresulting cells were concentrated using Negative Selection ColumnsGravity Feed Kit (StemSp) so that the ratio of CD8-positive T cellsbecame about 80%, and cocultured with the B-LCL cells pulsed with theWT1 peptide (fifth stimulation). CD8-positive T cells (CTLs) obtained 5days after the final stimulation were used for measurement of thecytotoxic activity.

Cytotoxic Activity of CTL

The cytotoxic activity of CTLs was measured using ⁵¹Cr release assay.CTL cells (hereinafter referred to as effector cells) were mixed at theratio (E/T ration) of 1:1 to 30:1 in 200 μl of medium with target cellsinto which ⁵¹Cr had been incorporated, and cultured in a 96-well cellculture plate at 37° C. with 5% CO₂ for 4 hours. B-LCL cells pulsed withthe same WT1 peptide as that used for induction of CTLs (BLCL-Ps), andB-LCL cells without pulsing with a WT1 peptide (BLCL-NPs) were used astarget cells. After the culture, the supernatants were collected bycentrifugation. The amounts of ⁵¹Cr released into the supernatants weremeasured using a liquid scintillation counter. The cytotoxic activity(%) was determined using the following formula:

(⁵¹Cr release in supernatant of sample−Spontaneous ⁵¹Crrelease)/(Maximum ⁵¹Cr release−Spontaneous ⁵¹Cr release)×100

wherein Spontaneous ⁵¹Cr release is ⁵¹Cr release observed when thetarget cells into which ⁵¹Cr had been incorporated were cultured aloneunder the same condition, and Maximum ⁵¹Cr release is ⁵¹Cr releaseobserved when the target cells into which ⁵¹Cr had been incorporatedwere completely lysed using 1% Triton X-100. Results are shown in FIGS.1-9. In the figures, longitudinal axes represent specific lysis (%), andhorizontal axes represent E/T ratios. BLCL-Ps are represented using fulllines, and BLCL-NPs are represented using dotted lines. It was confirmedthat CTLs induced with WT1₂₅₁, WT1₂₇₉, WT1₃₁₂, WT1₃₁₃, WT1₃₃₈, WT1₃₇₈WT1₃₈₆, WT1₄₁₅or WT1₄₃₆ damage specifically BLCL-Ps presenting the WT1peptide as a complex with an HLA-A*1101 molecule as compared withBLCL-NPs. CTLs induced with WT1₂₅₁, WT1₂₇₉, WT1₃₁₃, WT1₃₃₉ or WT1₃₈₆were used for additional experiments below.

Cytotoxic Activity of CTL Against Cell Expressing WT1 Endogenously

The cytotoxic activity of CTLs induced with WT1₂₅₁, WT1₂₇₉, WT1₃₁₃,WT1₃₃₈ or WT1₃₈₆ against B-LCLs expressing WT1 was determined using themethod described above. A cell expressing WT1 refers to a B-LCL intowhich a human WT1 gene is introduced, and that expresses a WT1 proteinin the cell, and presents a peptide consisting of about 9 amino acidsresulting from processing on an HLA-A*1101 molecule. Results are shownin FIGS. 1, 2, 4, 5 and 7. In the figures, B-LCLs expressing WT1 arerepresented using dashed lines. It was confirmed that CTLs induced withWT1₂₅₁, WT1₂₇₉, WT1₃₁₃, WT1₃₃₈ or WT1₃₈₆ have a cytotoxic activityagainst cells expressing WT1 gene endogenously.

Preparation of WT1 Peptide Dimer

A mixture of 227.5 mg of WT1₃₇₈ peptide monomer, 227.5 mg ofN-methylglucamine (NMG) and 23 ml of water was air-oxidized by stirringat room temperature for about 2 days. To the resulting mixture, anaqueous solution of 2 g of sodium acetate in 5 ml of water was added andthe mixture was stirred at room temperature for about 20 minutes. To theresulting solution, 200 ml of water and about 200 ml of acetonitrilewere added, and the mixture was filtered through Kiriyama funnel (filterpaper No. 5C) and washed with water (about 50 ml×3). To the residue,about 200 ml of water was added and the residue was lyophilized toobtain 158 mg of crude WT1₃₇₈ peptide dimer.

Purification of Crude WT1 Peptide Dimer

158 mg of the crude WT1₃₇₈ peptide dimer was dissolved in 9 ml of DMSOand injected into ODS C₁₈ column (5 cm Φ×50 cm L, YMC Co., LTD.)attached to HPLC (Shimadzu, LC8AD type) and equilibrated with solution 1(H₂O/1% AcOH) using a HPLC pump. The column was left for about 30minutes, and eluted with concentration gradient of 0% to 40% of solution2 (CH₃CN/1% AcOH) over 360 minutes. The fractions containing WT1 peptidedimer were collected using an automatic fraction collector whilemonitoring UV absorbance at 220 nm. The collected fractions werecombined, injected into ODS C₁₈ column (4.6 mm−×25 cm L, YMC Co., LTD.)attached to HPLC (Hitachi, L-4000 type) and equilibrated with 17% ofsolution 2, and eluted with concentration gradient of 0% to 47% ofsolution 2 over 30 minutes to obtain 46.6 mg of the purified WT1₃₇₈peptide dimer at retention time of 20.51 minutes.

FAB.MS 2365.0 (theoretical value: 2342.70) N⁺ F=0.25%

Induction of CTL by WT1 Peptide Dimer

Abilities of the resulting WT1₃₇₈ peptide dimer, WT1₃₇₈ peptide,modified WT1₃₇₈ peptide (G→I) (SEQ ID No: 11) and modified WT1₃₇₈peptide (G→V) (SEQ ID No: 12) as well as WT1₃₇₉ peptide (SEQ ID No: 13,as disclosed in WO 2002/28414) to induce a CTL were examined using PBMCsfrom HLA-A*1101-positive healthy donors 1-3 according to the method asdescribed above. Results are shown in FIGS. 10-14. In the figs,longitudinal axes represent specific lysis (%), and horizontal axesrepresent E/T ratios. BLCL-Ps are represented using full lines, andBLCL-NPs are represented using dotted lines. It was confirmed thatWT1₃₇₈ peptide dimer has an ability to induce a CTL. Furthermore, it wasfound that the ability of each WT1₃₇₉ peptide of which the amino acidsequence is different from that of WT1₃₇₈ peptide by one amino acid inthe amino acid sequence of WT1 protein to induce a CTL is much lowerthan that of WT1₃₇₈ peptide and, thus, the WT1 peptide of the presentinvention has an excellent and unexpected effect as compared with theknown peptide.

INDUSTRIAL APPLICABILITY

The present invention provides an HLA-A*1101-restricted WT1 peptide, apolynucleotide encoding the peptide, a pharmaceutical compositioncomprising the same and the like. Therefore, the present invention canbe used in the fields of medicine and the like, for example, in thefields of development and preparation of a pharmaceutical compositionfor the prevention or treatment of various hematopoietic tumors andsolid cancers that express WT1 gene at high levels.

Sequence Listing Free Text

SEQ ID NO: 11: Modified WT1 peptide

SEQ ID NO: 12: Modified WT1 peptide

1-19. (canceled)
 20. A method for the induction of a Wilms' tumor 1(WT1)-specific cytotoxic T cell (CTL), comprising administering to anHLA-A*1101-positive subject an effective amount of an isolated peptideconsisting of an amino acid sequence selected from: (SEQ ID No: 5)Ser Ala Ser Glu Thr Ser Glu Lys Arg; (SEQ ID No: 6)Ser His Leu Gln Met His Ser Arg Lys, and (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys;

and/or an isolated peptide dimer comprising a first and second peptidemonomer bound through a disulfide bond, wherein (1) the first peptidemonomer consists of an amino acid sequence Thr Gly Val Lys Pro Phe GinCys Lys (SEQ ID No: 7); and (2) the second peptide monomer consists ofan amino acid sequence selected from: (SEQ ID No: 3)Pro Ile Leu Cys Gly Ala Gln Tyr Arg, (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys, (SEQ ID No: 8)Lys Thr Cys Gln Arg Lys Phe Ser Arg, and (SEQ ID No: 9)Ser Cys Arg Trp Pro Ser Cys Gln Lys.


21. The method according to claim 20, wherein the isolated peptideconsists of Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ ID No: 7).
 22. AWT1-specific CTL, which is induced by an isolated peptide consisting ofan amino acid sequence selected from: (SEQ ID No: 5)Ser Ala Ser Glu Thr Ser Glu Lys Arg; (SEQ ID No: 6)Ser His Leu Gln Met His Ser Arg Lys, and (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys;

and/or an isolated peptide dimer comprising a first and second peptidemonomer bound through a disulfide bond, wherein (1) the first peptidemonomer consists of an amino acid sequence Thr Gly Val Lys Pro Phe GinCys Lys (SEQ ID No: 7); and (2) the second peptide monomer consists ofan amino acid sequence selected from: (SEQ ID No: 3)Pro Ile Leu Cys Gly Ala Gln Tyr Arg, (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys, (SEQ ID No: 8)Lys Thr Cys Gln Arg Lys Phe Ser Arg, and (SEQ ID No: 9)Ser Cys Arg Trp Pro Ser Cys Gln Lys.


23. A method for the induction of a WT1-specific CTL, comprisingculturing a peripheral blood mononuclear cell in the presence of anisolated peptide and/or an isolated peptide dimer to induce theWT1-specific CTL from the peripheral blood mononuclear cell, wherein theisolated peptide consists of an amino acid sequence selected from:(SEQ ID No: 5) Ser Ala Ser Glu Thr Ser Glu Lys Arg; (SEQ ID No: 6)Ser His Leu Gln Met His Ser Arg Lys, and (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys; and

wherein the isolated peptide dimer comprises a first and second peptidemonomer bound through a disulfide bond, wherein (1) the first peptidemonomer consists of an amino acid sequence Thr Gly Val Lys Pro Phe GinCys Lys (SEQ ID No: 7); and (2) the second peptide monomer consists ofan amino acid sequence selected from: (SEQ ID No: 3)Pro Ile Leu Cys Gly Ala Gln Tyr Arg, (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys, (SEQ ID No: 8)Lys Thr Cys Gln Arg Lys Phe Ser Arg, and (SEQ ID No: 9)Ser Cys Arg Trp Pro Ser Cys Gln Lys.


24. An antigen-presenting cell presenting a WT1 peptide, which isinduced by an isolated peptide consisting of an amino acid sequenceselected from: (SEQ ID No: 5) Ser Ala Ser Glu Thr Ser Glu Lys Arg;(SEQ ID No: 6) Ser His Leu Gln Met His Ser Arg Lys, and (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys;

and/or an isolated peptide dimer comprising a first and second peptidemonomer bound through a disulfide bond, wherein (1) the first peptidemonomer consists of an amino acid sequence Thr Gly Val Lys Pro Phe GinCys Lys (SEQ ID No: 7); and (2) the second peptide monomer consists ofan amino acid sequence selected from: (SEQ ID No: 3)Pro Ile Leu Cys Gly Ala Gln Tyr Arg, (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys, (SEQ ID No: 8)Lys Thr Cys Gln Arg Lys Phe Ser Arg, and (SEQ ID No: 9)Ser Cys Arg Trp Pro Ser Cys Gln Lys.


25. A method for the induction of an antigen-presenting cell presentinga WT1 peptide, comprising culturing an immature antigen-presenting cellin the presence of an isolated peptide and/or an isolated peptide dimerto induce the antigen-presenting cell presenting a WT1 peptide from theimmature antigen-presenting cell, wherein the isolated peptide consistsof an amino acid sequence selected from: (SEQ ID No: 5)Ser Ala Ser Glu Thr Ser Glu Lys Arg; (SEQ ID No: 6)Ser His Leu Gln Met His Ser Arg Lys, and (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys; and

wherein the isolated peptide dimer comprises a first and second peptidemonomer bound through a disulfide bond, wherein (1) the first peptidemonomer consists of an amino acid sequence Thr Gly Val Lys Pro Phe GinCys Lys (SEQ ID No: 7); and (2) the second peptide monomer consists ofan amino acid sequence selected from: (SEQ ID No: 3)Pro Ile Leu Cys Gly Ala Gln Tyr Arg, (SEQ ID No: 7)Thr Gly Val Lys Pro Phe Gln Cys Lys, (SEQ ID No: 8)Lys Thr Cys Gln Arg Lys Phe Ser Arg, and (SEQ ID No: 9)Ser Cys Arg Trp Pro Ser Cys Gln Lys.


26. A method for the diagnosis of a cancer, comprising incubating theCTL according to claim 22 with a sample from an HLA-A*1101-positivesubject, or administering the CTL to the HLA-A*1101-positive subject.27. The method according to claim 26, wherein the CTL is labeled.
 28. Amethod for the diagnosis of a cancer, comprising incubating theantigen-presenting cell according to claim 24 with a sample from anHLA-A*1101-positive subject, or administering the antigen-presentingcell to the HLA-A*1101-positive subject.
 29. The method according toclaim 28, wherein the antigen-presenting cell is labeled.